Evaluation of individual protein errors in silver-stained two-dimensional gels

• Published in: Biochemical and biophysical research communications Vol. 306; no. 4; pp. 1050 - 1055
• Main Authors: Mark Smales, C, Birch, John R, Racher, Andrew J, Marshall, Carol T, James, David C
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11-07-2003
• Subjects: 2-DE quantitation; Animals; Cell Line; Electrophoresis, Gel, Two-Dimensional - methods; Individual protein error; Mammalian cell culture; Mice; NSO proteome; Process error; Proteins - analysis; Reproducibility of Results; Silver stain; Silver Staining; 2-DE quantitation; NSO proteome; Mammalian cell culture; Silver stain; Process error; Individual protein error
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/S0006-291X(03)01115-X

The relationship between spot volume and variation for all protein spots observed on large format 2D gels when utilising silver stain technology and a model system based on mammalian NSO cell extracts is reported. By running multiple gels we have shown that the reproducibility of data generated in this way is dependent on individual protein spot volumes, which in turn are directly correlated with the coefficient of variation. The coefficients of variation across all observed protein spots were highest for low abundant proteins which are the primary contributors to process error, and lowest for more abundant proteins. Using the relationship between spot volume and coefficient of variation we show it is necessary to calculate variation for individual protein spot volumes. The inherent limitations of silver staining therefore mean that errors in individual protein spot volumes must be considered when assessing significant changes in protein spot volume and not global error.